Dilution pipette



Oct. 15, 1963 DIMMICK 3,106,845

DILUTION PIPETTE Filed June 26, 1961 2 Sheets-Sheet 1 rflzo INVENTOR.

Robert L. Dimmick BY m/J/J/ Oct. 15, 1963 R. L. DlMMlCK 3,406,845

DILUTION P'IPETTE Filed June 26, 1961 2 Sheets-Sheet 2 I II . W"? WU? Fig. 2.

v INVENTOR. Robert L. Dimmick BY [W W/Mm United States Patent 3,106,845 DHLUTIGN PEIPETTE Robert L. Dimmiclt, Albany, Qaliii, assignor to The Regents of the University of California, herlieiey, Calif.

Filed June 25, 1961, Ser. No. 119,437 12 Claims. (6!. 73-4256) This invention relates to the dilution of liquids. More particularly, it relates to a device for the rapid quantitative dilution of a sample solution with a suitable diluent such as in the dilution of a bacterial colloidal suspension with a nutrient liquid.

In a preferred embodiment the invention provides a cascading dilution pipette for diluting a sample solution to a mathematically determinable concentration comprising a plurality of chambers each having sides, a closed end, and an open end. An elongated hollow tip member is continuous with the closed end of each chamber and projects a preselected distance interiorly of the chamber. The chambers are removably joined serially in open end to close end fluid tight relation with the tip members establishing fluid communication with the adjacent chambers.

Liquid moving means is associated with the tip member of the initial chamber. A closure seals the open end of the terminal chamber and a hollow elongated tip member is associated with the closure establishing fluid communication exteriorly of the terminal chamber. The liquid moving means is operable to serially fill selected chambers up to the end of their associated tip members with a diluent liquid when the chambers are in a generally vertical position with the ends of the tip members pointing generally downwardly.

The size and shape of the chambers and the length and position of the tip members are such that when the chambers are filled up to the ends of the tip members in a generally vertical position and then shifted to a relatively horizontal position, the liquid level in the chambers is below the open ends of the tip members. As a result, the initial chamber may be removed and a sample to be diluted placed therein without altering the quantity of fluid present in any of the liquid containing chambers since the diluent :present is unaifected by pressure changes.

After the sample solution has been placed in the initial chamber, the chamber returned to its former position, and the entire device placed in the generally vertical position with the ends of the tip members pointing generally downwardly, the fluid moving means, chambers, and tip members are cooperably operable to transfer a preselected portion of the liquid in a chamber to the adjacent chamber and eXteriorly of the terminal chamber.

Since the volume of diluent in each chamber is known, the concentration of the known quantity of transferred liquid in any given chamber is easily calculated. Transferof sample solution to the next adjacent chamber from the initial chamber results in a diluted sample solution. By repeating the transfer of known amounts of solution serially to adjacent chambers, dilution to the extent desired is obtained.

As used throughout this specification and claims, the terms solution and liquid are used in the broadest sense and include true solutions as well as for example, colloidal suspensions.

Reference is made to the accompanying drawings, in which:

FIG. 1 shows in side section a dilution pipette in a vertical position in accordance with the present invention in which all but the initial chamber contains diluent.

FIG. 2 shows in side elevation the position of the liquid in the chambers of FIG. 1 when the pipette is placed in the generally horizontal position.

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FIG. 3 shows in perspective a single chamber of the device of FIGS. 1 and 2.

FIG. 4 shows in side section a single chamber for a device in accordance with the present invention in which the internal hollow tip member is in a preferred position and in which a bafile has been added to the chamber.

FIG. 5 shows in side elevation an alternate embodiment for the present invention in which the chambers are in the form of a funnel.

The principal advantage of the present device is in the ease and rapidity with which accurate dilutions may be made without the necessity of elaborate or expensive equipment. This advantage is best appreciated when the prior art methods are first considered.

As an example, in the biological arts, in order to make a particulate count of highly concentrated collodial suspensions it has been necessary to dilute the suspensions in a piecemeal, time-consuming manner. The necessity for dilution arises since it is not uncommon to deal with bacterial suspensions .which contain as many as a thousand billion organisms per fluid milliliter. The highest number of bacteria which can be accurately counted in the form of colonies on nutrient agar plates is about 300. Thus, an 8 to 10-fold decimal dilution procedure is required to provide suspensions which can be counted. The same orders of magnitude are involved in virology studies.

To make the dilution, prior practice has been to employ a plurality of test tubes and pipettes, all requiring prior sterilization and special handling. One test tube contains the material to be diluted and the remaining test tubes contain diluent as required depending on the degree of dilution sought. The procedure is to pipette a known amount from the sample to the first diluent containing test tube, thoroughly disperse the sample in the diluent, remove a portion of the first dilution product and place it in a second tube containing diluent, and so forth until the desired dilution is obtained.

'Ihis prior conventional method requires a great deal of time both in carrying out the dilution itself and in cleaning the large amount of equipment required. There is also a great possibility of introducing contamination during the many repeated handling steps. The present invention eliminates all of the numerous handling steps and time-consuming preparation. It involves only one instrument for the entire dilution operation and is extremely rapid in comparison with the prior procedure.

Referring to the drawings, it will be seen that the present invention provides in a preferred embodiment a cascading dilution pipette in which the individualcontainer or chamber 16 has the form of a cylindrical cake pan of the type having a hollow frusto conical member l1 projecting inwardly from the bottom. Frusto conical member 11 serves as the elongated hollow tip member referred to hereinbefore. The lower end of member 11 forms an orifice 32. T he chambers iii are assembled open end 13 to closed end 14 with the closed end 14 of the first or initial chamber it) marked A being one end or closure of the series of chambers.

As assembled, orifice or opening 12 in member 11 serves to establish fluid communication between adjacent chambers. The chambers 10 are removably joined in fluid tight relation by means of stepped lip 15 at the open end 13 of chamber in. As may be seen for example in FIG. 1, when the chamber marked A is brought into contact with the chamber marked B with the open end 13 of A abutting the closed end 14 of B, the lip 15 of chamber A engages, in liquid tight relation, the Walls 16 of chamber B in the vicinity of closed end 14-. Chamber A may be disconnected from chamber B merely by pulling them apart.

Positioned on the initial chamber A is a rubber syringe 17 which is in fluid communication with the interior of chamber A through its tip member 11 and an extension tube 13 therefor. The terminal chamber or chamber D includes a fluid tight closure 19 for its open end 13. Closure 19 is removably attached to lip 15 of chamber D in the same way that the closed end 14 of chamber B is joined to lip 15 of chamber A. Closure 19 has a depending tip member 11' and an orifice 12 therein similar to tip members 11 described above which communicates exteriorly of chamber D.

With reference to FIG. 1, the pipette is filled with diluent by holding the entire device in the generally vertical position as shown with the tip members 11, 11 pointing generally downwardly. The tip member 11' which communicates exteriorly of chamber D is inserted in a container 26 containing a diluent 21. By positioning syringe 17 so that a negative pressure is created throughout all of the chambers, the liquid or diluent 21 will be drawn up through the tips 11, 11 serially until the negative pressure is equilibrated. At that point the level of the fluid 21 in chambers is fixed. ln P16. 1 the negative pressure has been equilibrated after the liquid 21 has been drawn just to but not including chamber A. It is noted that the fluid in each chamber rises only to the bottom of its tip member 11 when the device is held in the generally vertical position before the next upper adjacent chamber is filled. The volumes contained in each chamber are predetermined before using the device for its intended purpose.

To place a sample to be diluted in chamber A, the entire device is moved to a generally horizontal position as shown in FIG. 2. The liquid 21 in each chamber is now below the orifices 12 and is out of contact therewith. As a result chamber A may be disengaged from chamber B by sliding lip of chamber A out of engagement with closed end 14 of chamber B without altering the fluid volume in any of the fluid containing chambers. Syringe 17 may be removed from chamber A by sliding it away from extension tube 13.

Chamber A is suitably inverted with the open end 13 pointing upwardly. A sample to be diluted is placed inside chamber A in an amount so that the liquid level is below the orifice 12 of tip 11. Chamber A is then replaced on chamber B as noted before while maintaining the entire unit in the generally inverted position with open ends 13 facing upwardly. Again, diluent 21 will not contact orifices 12 and equilibrium pressure conditions continue to exist. Syringe 17 may then, if it has been removed, be replaced on tube 18 and the entire unit is moved back to a generally vertical position whereby the fluid 21 in each chamber fills the tip 11 of the next lower adjacent chamber and tip 11 of terminal chamber D.

By positioning syringe 17 so that a positive pressure is created throughout all of the chambers, a volume of the liquid in each chamber is transferred to the next adjacent chamber. This volume is controlled by the amount of positive pressure exerted or created by syringe 17. Volume control is best accomplished when orifices 12 do not contact the fluid 21 in their own chambers. This is possible if positive pressure is applied while maintaining the unit in other than a perfectly vertical position. Such a precaution avoids forming one continuous column of fluid and allows the transfer of as little as one drop at a time.

Preferably all of the orifices 12 of tips 11, 11' have equal diameters so that a constant volume is transferred from chamber to chamber. However, it will be apprecited by those skilled in the art that the volume flowing through any tip 11, 11' or the volumes contained in any of the chambers may be varied to cause greater or lesser dilution to occur in any chamber and these differences may be accounted for in a final calculation to determine the dilution that has occurred at a given time.

The known controlled quantity of fluid is transferred from chamber to chamber because of the concurrent pressure exerted throughout the system when the positive pressure from syringe 17 is exerted on chamber A which in turn transmits the pressure to chamber B and so on throughout the chambers. A known volume is discharged exteriorly through tip 11 of chamber D as a result of the chain-like reaction.

The volume transferred to the chambers B, C, and D from the precedin chambers respectively, is suitably thoroughly and uniformly dispersed by again placing the unit in the generally horizontal position shown in FIG. 2 and agitating the fluid by motion at a right angle to the longitudinal axis of the device. This tends to prevent unwanted passage of fluid during the agitation period.

To further prevent any unwanted passage of fluid, and generally for ease of operation and control of transfer portion size, the tip member 11 may be positioned adjacent to the side 16 of the chamber 10 as shown in FIG. 4. Also as an aid in obtaining uniform dispersion of the transferred liquid in the liquid already present, a baflie 22 as shown in FIG. 4 may be included in the chambers 10, suitably attached to the side 16 for example.

By repeating the transfer of a known quantity of fluid serially starting from chamber A on through chamber D and exteriorly thereof, causing the transferred material to first be uniformly dispersed in the receiving chamber before subsequent transfers are made, any given dilution may be obtained in the terminal or intermediate chambers. The more portions transferred, the greater the sample solution concentration in a given chamber after the first transferred portion containing some sample solution from chamber A is received.

It is noted that the relation of chamber size, position of tip member 11 in the chamber 10, and the distance that tip member 11 extends interiorly of chamber 10 is such that when liquid is present in chamber 10 up to the bottom of tip member 11 in the generally vertical position, the chamber may be rotated either generally horizontally or inverted entirely so that the fluid in the container does not contact the open end of tip member 11 and enter orifice 12. Preferably this configuration will be such that tip member 11 extends more than half way into chamber 10 so that the air space in chamber 10, when liquid is present up to the end of tip member 11, is greater than the liquid volume. Shifting the position of tip member 11 to one side as in FIG. 4 further facilitates the accomplishment of this object. It will be obvious that numerous other configurations will similarly accomplish the desired result.

In the alternate embodiment shown in FIG. 5, the principle of operation and construction is similar to that described above except that the chambers 10 are in the form of funnel sections. Suitably the funnel sections taper in diameter from larger to smaller proceeding from the initial chamber to the terminal chamber. Since it is preferable to have constant volume chambers, the chambers may be increased in height to compensate for the gradual reduction in diameter. This has the further advantage in permitting turning of the unit into the horizontal or inverted positions while more positively avoiding liquid contact with the orifice 12 of tip member 11. This advantage will be appreciated when it is considered that during the operation of the device, the volume decreases in the initial and immediately following chambers while it increases in the chambers near and at the terminal end. This tapered-increasing height configuration is well adapted to handle the increased volume in the terminal and its nearby chambers during rotation of the device.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is understood that certain changes and modifications may be practiced within the spirit of the invention as limited only by the scope of the appended claims.

What is claimed is:

1. A system for rapidly diluting solutions including a plurality of means for holding a plurality of predetermined quantities of liquid, means for establishing serial fluid communication between the liquid holding means, the adjacent liquid holding means in the communication series having a common wall through which serial communication is established, discharge means cooperating with the terminal liquid holding means permitting external discharge therefrom, fluid moving means cooperat ing with all of said liquid holding means and said fluid communication establishing means to self-meterably fill selected ones of said liquid holding means with predetermined quantities of liquid from an external source, means for introducing a sample liquid to be diluted into at least one of said liquid holding means, said fluid moving means being alternatively operable for controllably moving a preselected portion of liquid in each holding means serially through said fluid communication establishing means from each holding means to the serially adjacent holding means and exteriorly from said terminal holding means, whereby the concentration of sampie liquid in each liquid holding means may be mathematically calculated.

2. A dilution device comprising a plurality of chambers each adapted for holding a predetermined quantity of liquid, said chambers being serially interconnected in a fluid tight manner, the adjacent chambers in the interconnected series having a common wall, a fluid communication conduit between adjacent chambers in said common wall therebetween and from the terminal chamber to the exterior thereof, fluid moving means cooperating with the chambers and conduits to draw fluid in from an exterior source through the terminal chamber and to serially deposit a predetermined quantity of the fluid in each of a selected number of said chambers, said chambers and conduits having a configuration permitting at least one chamber to receive a sample of liquid to be diluted without altering the quantity of fluid present in any of the other chambers, and said fluid moving means, chambers, and conduits being cooperably operable to controllably transfer a preselected portion of the liquid present in a chamber to the adjacent chamber and to the exterior of the terminal chamber, whereby the concentration of sample liquid in a chamber may be mathematically calculated.

3. A dilution pipette for diluting a sample solution to a mathematically determinable concentration comprising a piurality of chambers each having a preselected volume serially joined in fluid tight relation, an elongated hollow tip member continuous with one end of each chamber projecting a preselected distance interiorly of the chamber to establish fluid communication with the adjacent chamber, all of said tip members being substantially unidirectionally oriented, liquid moving means associated with the initial chamber, a hollow elongated tip member projecting outwardly of the terminal chamber establishing fluid communication exteriorly thereof, said liquid moving means operable to serially fill selected chambers up to the end of their associated tip members when the chambers are in a generally vertical position with the ends of the tip members pointing generally downwardly, said chambers of suflicient size to contain said vertical position filling amount of liquid therein out of fluid communication with the end of the tip member therein when said chambers are moved from vertical position so that at least one chamber may receive a sample of liquid to be diluted without altering the quantity of fluid present in any of the other chambers, and said fluid moving means, chambers, and tip members being cooperably operable to transfer a preselected portion of liquid in any chamber to the adjacent chamber and exteriorly of the terminal chamber. a

, 4. A dilution pipette in accordance with claim 3 wherein the chambers and tip members are adapted to permit agitation of liquid in the chambers to uniformly disperse d a transferred portion of liquid in an adjacent chamber without altering the quantity of fluid present in any of the chambers.

5. A cascading dilution pipette for diluting a sample solution to a mathematically determinable concentration comprising a plurality of chambers each having sides, a closed end, and an open end, an elongated hollow tip member continuous with the closed end of each chamber projecting a preselected distance interiorly of the charnber, said chambers being removably joined serially in open end to closed end fluid tight relation with said tip members establishing fluid communication with the adjacent chamber, liquid moving means removably connected with the tip member of the initial chamber, a closure for the open end of the terminal chamber, a hollow elongated tip member projecting outwardly of said closure establishing fluid communication exteriorly of said terminal chamber, said liquid moving means operable to serially fill selected chambers up to the end of their as sociated tip members when the chambers are in a gencrally vertical position with the ends of the tip members pointing generally downwardly, the size and shape of the chambers and the length and position of the tip members therein being such that when the chambers are filled up to the ends of the tip members in a generally vertical position and then shifted to a relatively horizontal position, the liquid level is below the ends of the tip members whereby a liquid sample to be diluted may be inserted in a removed liquid-free chamber without altering the quantity of fluid present in any of the liquid containing chambers, said fluid moving means, chambers, and tip members being cooperably operable when in the general vertical position with the ends of the tip members pointing generally downwardly to transfer a preselected portion of the liquid in a chamber to the adjacent lower chamber and exteriorly from the terminal chamber.

6. A dilution pipette in accordance with claim 5 wherein the tip members extend a sufficient distance within each chamber so that when filled with liquid to the end of the tip in the generally vertical position, the air volume in the chamber is greater than the liquid volume in the chamber. v

7. A dilution pipette in accordance with claim 5 wherein each chamber has a baflie member fixed to a side thereof for promoting uniform dispersion of a transferred portion of liquid in the chamber.

8. A cascading dilution pipette for diluting a sample solution to a mathematically determinable concentration comprising a plurality of chambers each having a preselected volume serially disconnectably joined in end to end fluid tight relation, an elongated hollow tip member continuous with one end of each chamber projecting a preselected distance interiorly of the chamber and adjacent to one side thereof to establish fluid communication with an adjacent chamber, all of said tip members being substantially unidirectionally oriented, a rubber syringe in fluid communication with the initial chamber for creating positive and negative pressures throughout all of the chambers, a hollow elongated tip member projecting outwardly of the terminal chamber establishing fluid communication exteriorly thereof, whereby when said syringe is positioned to create a negative pressure within the chambers when the chambers are in a generally vertical position with the ends of the tip members pointing genera-l ly downwardly, said chambers may be serially filled with liquid up to the end of its associated tip member starting with the terminal chamber and ending with a preselected chamber, said chamber of suflicient size to contain said vertical position filling amount of. liquid therein out of fluid communication with the end of the tip members therein when said chambers are moved from vertical position so that a disconnected liquid-free chamber may receive a sample of liquid .to be diluted without altering the quantity of fluid present in any of the other chambers, and whereby when the syringe is positioned to create a controlled positive pressure within the chambers when the chambers are in a generally vertical position with the ends of the tip members pointing generally downwardly, a drop of the liquid present in a chamber is forced into the adjacent chamber and exteriorly from the terminal chamber.

9. A dilution pipette in accordance with claim 8 wherein the volume of each chamber between the end of the tip member and the opposite end of the chamber is substantially equal.

10. A dilution pipette in accordance with claim 9 wherein the internal diameter of the end of each hollow tip member is substantially equal so that when said syringe is positioned to create a positive pressure, the volume of liquid serially transferred through each tip member is substantially uniform.

11. A dilution pipette in accordance with claim 10 wherein each chamber has the form of a cylindrical cake pan of the type having a hollow frusto-conical member projecting inwardly from its bottom.

12. Apparatus for serially diluting a solution in known increments comprising, in combination, a plurality of containers, means for connecting said containers in a serial array, each of said containers having a fluid orifice providing fluid communication with the adjacent container of said serial array, a control orifice formed in the first container of said array, the last container of said array provided with a terminal orifice establishing fluid communication exteriorally thereof, a source of diluent, the first container in said array provided with means for drawing known quantities of said diluent through said terminal orifice and selected ones of the fluid communicating orifices between adjacent containers preparatory to discharging a metered quantity of said diluent therefrom into the adjacent containers, means for introducing a quantity of solution to be diluted into said first container of the array, said apparatus upon the discharging of a metered quantity of solution into the next adjacent container causing concurrent discharges of metered quantities of the solution and diluent in each container through the fluid communicating. orifices between adjacent containers and the terminal orifice associated with the last container in said array.

Hapgood Apr. 14, 1936 Chenet-te May 27, 1958 

1. A SYSTEM FOR RAPIDLY DILUTING SOLUTIONS INCLUDING A PLURALITY OF MEANS FOR HOLDING A PLURALITY OF PREDETERMINED QUANTITIES OF LIQUID, MEANS FOR ESTABLISHING SERIAL FLUID COMMUNICATION BETWEEN THE LIQUID HOLDING MEANS, THE ADJACENT LIQUID HOLDING MEANS IN THE COMMUNICATION SERIES HAVING A COMMON WALL THROUGH WHICH SERIAL COMMUNICATION IS ESTABLISHED, DISCHARGE MEANS COOPERATING WITH THE TERMINAL LIQUID HOLDING MEANS PERMITTING EXTERNAL DISCHARGE THEREFROM, FLUID MOVING MEANS COOPERATING WITH ALL OF SAID LIQUID HOLDING MEANS AND SAID FLUID COMMUNICATION ESTABLISIHING MEANS TO SELF-METERABLY FILL SELECTED ONES OF SAID LIQUID HOLDING MEANS WITH PREDETERMINED QUANTITIES OF LIQUID FROM AN EXTERNAL SOURCE, MEANS FOR INTRODUCING A SAMPLE LIQUID TO BE DILUTED INTO AT LEAST ONE OF SAID LIQUID HOLDING MEANS, SAID FLUID MOVING MEANS BEING ALTERNATIVELY OPERABLE FOR CONTROLLABLY MOVING A PRESELECTED PORTION OF LIQUID IN EACH HOLDING MEANS SERIALLY THROUGH MEANS TO THE SERIALLY ADJACENT MEANS FROM EACH HOLDING MEANS TO THE SERIALLY ADJACENT HOLDING MEANS AND EXTERIORLY FROM SAID TERMINAL HOLDING MEANS, WHEREBY THE CONCENTRATION OF SAMPLE LIQUID IN EACH LIQUID HOLDING MEANS MAY BE MATHEMATICALLY CALCULATED. 